Gene expression patterns of Helicoverpa armigera gut proteases (original) (raw)
Related papers
2006
Protease inhibitors mediate a natural form of plant defence against insects, by interfering with the digestive system of the insect. In this paper, affinity chromatography was used to isolate trypsins and chymotrypsins from Helicoverpa zea larvae, which had been raised on inhibitor-containing diet. Sensitivity of the fractions to inhibition by plant proteinase inhibitors was tested, and compared to the sensitivity of proteinases found in insects raised on diet to which no inhibitor had been added. The isolated chymotrypsin activity was found to be less sensitive to plant protease inhibitors. The sensitivity of the isolated trypsin activity was found to be intermediate between completely sensitive trypsins and completely insensitive forms that have been previously described. Mass spectrometry was used to identify one trypsin and two chymotrypsins in the partially purified protease fraction. The sequence features of these proteases are discussed in relation to their sensitivity to inhibitors. The results provide insight in the enzymes deployed by Helicoverpa larvae to overcome plant defence.
Journal of Chromatography B-analytical Technologies in The Biomedical and Life Sciences, 2006
Protease inhibitors mediate a natural form of plant defence against insects, by interfering with the digestive system of the insect. In this paper, affinity chromatography was used to isolate trypsins and chymotrypsins from Helicoverpa zea larvae, which had been raised on inhibitor-containing diet. Sensitivity of the fractions to inhibition by plant proteinase inhibitors was tested, and compared to the sensitivity of proteinases found in insects raised on diet to which no inhibitor had been added. The isolated chymotrypsin activity was found to be less sensitive to plant protease inhibitors. The sensitivity of the isolated trypsin activity was found to be intermediate between completely sensitive trypsins and completely insensitive forms that have been previously described. Mass spectrometry was used to identify one trypsin and two chymotrypsins in the partially purified protease fraction. The sequence features of these proteases are discussed in relation to their sensitivity to inhibitors. The results provide insight in the enzymes deployed by Helicoverpa larvae to overcome plant defence.
Journal of Insect Physiology, 2010
We evaluated 22 different host and non-host plant protease inhibitors (PIs) for in vivo inhibition of Helicoverpa armigera gut pro-and proteinases, and their biological activity against the pod borer, H. armigera, the most important pest of agriculture and horticultural crops worldwide. In vitro activation of H. armigera gut pro-proteinases (HaGPPs) in larvae fed on non-host plant PIs showed significant in vivo inhibition of HaGPPs activation in solution as well as in gel assays. The larvae fed on diet incorporated with Datura alba ness PIs showed highest inhibition of HaGPPs, followed by Psophocarpus tetragonolobus. Non-host plant PIs from Pongamia pinnata, Mucuna pruriens, Capsicum annuum, and Nigela sativa showed maximum inhibitory potential towards HaGPs in vivo, and also exhibited moderate level of inhibition of pro-proteinases. However, some of non-host plant PIs, such as those from Penganum harmala and Solanum nigrum, and the principal host plant PIs, viz., Cicer arietinum and Cajanus cajan did not inhibit HaGPP activity. Pro-proteinase level increased with the growth of the larvae, and maximum HaGPP activity was observed in the fifth-instars. Larvae fed on diets with D. alba ness PIs showed greater inhibition of HaGPPs as compared to the larvae fed on diets with P. tetragonolobus. Low concentrations of partially purified HaGPs treated with gut extract of larvae fed on D. alba ness showed that out of 10 proteinase isoforms, HaGPs 5 and 9 were activators of pro-proteinases. Larval growth and development were significantly reduced in the larvae fed on the non-host plant PIs, of which D. alba ness resulted in highest stunted growth of H. armigera larvae. The in vivo studies indicated that non-host plant PIs were good candidates as inhibitors of the HaGPs as well as HaGPPs. The PIs from the non-host plants can be expressed in genetically engineered plants to confer resistance to H. armigera. ß
Plant Physiology, 1999
We report on the efficacy of proteinase inhibitors (PIs) from three host plants (chickpea [Cicer arietinum], pigeonpea [Cajanus cajan], and cotton [Gossypium arboreum]) and three non-host (groundnut [Arachis hypogea], winged bean [Psophocarpus tetragonolobus], and potato [Solanum tuberosum]) in retarding the growth ofHelicoverpa armigera larvae, a devastating pest of important crop plants. Enzyme assays and electrophoretic analysis of interaction of H. armigera gut proteinases (HGPs) with PIs revealed that non-host PIs inhibited HGP activity efficiently whereas host PIs were ineffective. In the electrophoretic assay, trypsin inhibitor activity bands were detected in all of the host and non-host plants, but HGP inhibitor activity bands were present only in non-host plants (except cotton in the host plant group). H. armigera larvae reared on a diet containing non-host PIs showed growth retardation, a reduction in total and trypsin-like proteinase activity, and the production of inhibi...
Frontiers in Physiology, 2016
Proteinase inhibitors (PIs) are natural defense proteins of plants found to be active against gut proteases of various insects. A pigeonpea wild relative Cajanus platycarpus was identified as a source of resistance against Helicoverpa armigera, a most devastating pest of several crops including pigeonpea. In the light of earlier studies, trypsin-specific PIs (CpPI 63) were purified from mature dry seeds of C. platycarpus (ICPW-63) and characterized their biochemical properties in contributing to H. armigera resistance. CpPI 63 possessed significant H. armigera gut trypsin-like proteinase inhibitor (HGPI) activity than trypsin inhibitor (TI) activity. Analysis of CpPI 63 using two-dimensional (2-D) electrophoresis and matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry revealed that it contained several isoinhibitors and small oligomers with masses ranging between 6 and 58 kDa. The gelatin activity staining studies suggest that these isoinhibitors...
Insect Biochemistry and Molecular Biology, 2001
Helicoverpa armigera is a devastating pest of cotton and other important crop plants all over the world. A detailed biochemical investigation of H. armigera gut proteinases is essential for planning effective proteinase inhibitor (PI)-based strategies to counter the insect infestation. In this study, we report the complexity of gut proteinase composition of H. armigera fed on four different host plants, viz. chickpea, pigeonpea, cotton and okra, and during larval development. H. armigera fed on chickpea showed more than 2.5-to 3-fold proteinase activity than those fed on the other host plants. H. armigera gut proteinase composition revealed the predominance of serine proteinase activity; however, the larvae fed on pigeonpea revealed the presence of metalloproteases and low levels of aspartic and cysteine proteases as well. Gut proteinase activity increased during larval development with the highest activity seen in the fifth instar larvae which, however, declined sharply in the sixth instar. Over 90% of the gut proteinase activity of the fifth instar larvae was of the serine proteinase type, however, the second instar larvae showed the presence of proteinases of other mechanistic classes like metalloproteases, aspartic and cysteine proteases along with serine proteinase activity as evident by inhibition studies. Analysis of fecal matter of larvae showed significant increase in proteinase activity when fed on an artificial diet with or without non-host PIs than larvae fed on a natural diet. The diversity in the proteinase activity observed in H. armigera gut and the flexibility in their expression during developmental stages and depending upon the diet provides a base for selection of proper PIs for insect resistance in transgenic crop plants.
Process Biochemistry, 2014
A protease inhibitor from the seeds of Butea monosperma (BmPI) was purified, characterized and studied for its influence on developmental physiology of Helicoverpa armigera. BmPI on two-dimensional separations indicated the presence of a 14 kDa protein with an isoelectric point in the acidic region (pI 5.6). Multiple Sequence Analysis data suggested that the BmPI contains a sequence motif which is conserved in various trypsin and chymotrypsin inhibitors of Kunitz-type. The inhibitor exhibited trypsin inhibitory activity in a broad range of pH (4-10) and temperature (10-80 • C). The enzyme kinetic studies revealed BmPI as a competitive inhibitor with a K i value of 1.2 × 10 −9 M.
Proteomics, 2010
Six diverse representative Capsicum annuum (common name: hot pepper; Solanaceae) protease inhibitor genes, viz CanPI-5, -7, -13, -15, -19, and 22 comprising 1–4 inhibitory repeat domains (IRDs), were cloned and expressed in Pichia pastoris. The recombinant proteins were evaluated for their interactions with bovine trypsin, chymotrypsin, and Helicoverpa armigera gut proteases (HGP) using electrophoretic (native and denaturing) and mass spectrometric (MALDI-TOF-MS in combination with intensity fading assays) techniques. These techniques allow qualitative and semiquantitative analysis of multiple and processed IRDs of purified recombinant Capsicum annuum proteinase inhibitor (rCanPI) proteins. rCanPIs showed over 90% trypsin inhibition, varying chymotrypsin inhibition depending on the number of respective IRDs and over 60% inhibition of total HGP. rCanPI-15 that has only one IRD showed exceptionally low inhibition of these proteases. Interaction studies of rCanPIs with proteases using intensity fading-MALDI-TOF-MS revealed gradual processing of multi-IRD rCanPIs into single IRD forms by the action of HGP at the linker region, unlike their interactions with trypsin and chymotrypsin. Intensity fading-MALDI-TOF-MS assay showed that CanPI-13 and -15, possessing single IRD and expressed predominantly in stem tissue are degraded by HGP; indicating their function other than defense. In vitro and in vivo studies on rCanPI-5 and -7 showed maximum inhibition of HGP isoforms and their processed IRDs were also found to be stable in the presence of HGP. Even single amino acid variations in IRDs were found to change the HGP specificity like in the case of HGP-8 inhibited only by IRD-12. The presence of active PI in insect gut might be responsible for changed HGP profile. rCanPI-5 and -7 enhanced HGP-7, reduced HGP-4, -5, -10 expression and new protease isoforms were induced. These results signify isoform complexity in plant PIs and insect proteases.
American Journal of Plant Sciences, 2012
Cotton bollworm/legume pod borer, Helicoverpa armigera is one of the most damaging pests worldwide. Because of the difficulties associated with chemical control of this pest, emphasis has been placed on developing transgenic plants with resistance to H. armigera. Since toxin genes from the bacterium, Bacillus thuringiensis (Bt) have been deployed on a large scale, there is need to scout for alternate genes which could be deployed alone or in combination with the Bt genes for pest management. Therefore, we evaluated the wild relatives of pigeonpea, which have shown high levels of resistance to this pest, for the protease inhibitors (PIs) under in vivo and in vitro inhibitions. Accessions belonging to Cajanus albicans, C. cajanifolius, C. sericeus, Flemingia bracteata, and Rhynchosia bracteata showed complete inhibition of H. armigera gut proteinases (HaGPs). Some of the C. scarabaeoides accessions (ICPW 116, 152, 278 and 280) exhibited partial inhibition at low concentrations of the PIs. All accessions of wild relatives of pigeonpea showed high to moderate level of inhibition at pH 7.8. Cultivated pigeonpea, ICPL 87 exhibited monomorphism in terms of trypsin inhibitor (TI) and chymotrypsin inhibitor (CTI) isoforms, contrary to the diverse inhibitory profiles of wild pigeonpeas. Cajanus albicans, C. platycarpus, C. scarabaeoides, and R. bracteata showed more number of TI and CTI bands than the cultivated pigeonpea. Protease inhibitor isoforms of wild relatives of pigeonpea showed significant variation in number, band pattern, and protein specificities towards trypsin, chymotrypsin, and H. armigera gut proteinases (HaGPs) as compared to the cultivated pigeonpea. The PIs from the wild relatives of pigeonpea showed considerable potential against the HaGPs, and could be considered as potential candidates for use in genetic transformation of crops for pest management, including H. armigera.
European Journal of …, 2005
Membrane-bound proteases from preparations of the midgut of 5 th instar velvetbean caterpillars, Anticarsia gemmatalis (Hübner) were obtained by resuspension of the pellet obtained after 100,000 g centrifugation. As expected of trypsin-like proteases, they hydrolyzed casein and the synthetic substrates N--benzoyl-L-Arg-p-nitroanilidine (L-BApNA) and N--p-tosyl-L-Arg methyl ester (L-TAME). Higher activities were observed at 50°C, and at pH 8.5 and 8.0 for both synthetic substrates L-BApNA and L-TAME. The membrane-bound proteases were inhibited by EDTA, phenylmethan sulphonyl fluoride (PMSF), tosyl-L-lysine chloromethyl ketone (TLCK), benzamidine and aprotinin. TLCK and benzamidine were particularly active inhibitors. The KM-values obtained were 0.23 mM for L-BApNA and 92.5 µM for L-TAME. These results provide evidence for the presence of membranebound trypsin-like proteases in the midgut of the velvetbean caterpillar, a key soybean pest in warm climates. The interaction between A. gemmatalis digestive proteases and soybean protease inhibitors has potentially important consequences for soybean breeding programs.